Covalent conjugation of biochemical molecules can be employed to bring together two or more molecules to form a bioconjugate that displays the combined properties of each of the individual components. This technique has been used to increase plasma half-life and decrease immunogenicity of therapeutic agents, such as peptides. Typically, the therapeutic agent is conjugated to a macromolecular carrier directly or via a linker. Common macromolecular carriers include antibodies, albumin and synthetic polymers.
U.S. Pat. Nos. 7,521,425 and 8,288,349 describe processes for preparing compounds useful as linkers.
The reference to any art in this specification is not, and should not be taken as, an acknowledgement of any form or suggestion that the referenced art forms part of the common general knowledge.
Background of Conjugation Process
Antibody-drug conjugate 5 has been described in U.S. Pat. No. 8,288,349 (whose contents are hereby incorporated entirely), the production of which involves several stages. Initially, the peptide 2 and the linker 1 are prepared separately. The peptide 2 is then conjugated to the linker 1 to form the linker-peptide complex (3). After purification, the conjugated linker-peptide complex 3 is combined with the antibody (4) so as to allow the azetidinone moiety of 3 form a covalent bond with the antibody 4, thereby resulting in an assembled peptide-linker-antibody complex; the antibody-drug conjugate 5 (scheme I). The linker peptide complex is prepared by a lengthy multi-step process requiring generation of the linker 1, 8 and conjugation to the peptide 2 (scheme II).
The peptide is shown schematically, with the available lysine side-chain indicated. The lysine residue forming the covalent bond with the linker is preferably the only unmodified lysine in the peptide, to avoid multiple species forming. The lysine may be located as the N-terminal residue, the C-terminal residue, or anywhere within the peptide chain (for example SEQ ID NOs:1 and 2). The antibody is also shown schematically, with the reactive side chain indicated. Typically, the antibody is a catalytic antibody such as an aldolase catalytic antibody comprising a reactive lysine in the antibody combining site (antigen recognition site, or CDR), as further described herein and also in U.S. Pat. Nos. 7,521,425, 8,288,349, and 8,252,902.
Synthesis of Acid Linker 6
The original synthesis of acid 6 is shown in scheme II. Acid 9 is treated with Cl2SO at reflux to provide crude acid chloride 10. In a separate flask, 2-azetidinone 11 is deprotonated at cryogenic temperature with n-BuLi in THF to generate Li anion 12 which, without isolation, is reacted with acid chloride 10 to give intermediate 13 in 91% yield. The nitro group on 13 is then reduced using catalytic hydrogenation with 10% Pd/C in MeOH to give aniline HCl salt 14b in 68% yield. The last step involves the reaction between 14b and diglycolic anhydride (15) in CH2Cl2 in the presence of DIPEA to provide acid 6 in 83% yield.
During preliminary experiments to determine the scalability of scheme II, it was found that the yield for the coupling between acid chloride 10 and azetidinone 11 was not reproducible and dropped to about 40-55% when the reaction was run on about 200-g scale. There therefore exists a need to find an alternative mechanism to generate acid 6 with the additional goal of avoiding running the process at cryogenic temperature.
Synthesis of Peptide-Linker 3
Acid 6 and N-hydroxysuccinimide (7) undergo reaction with N,N′-diisopropylcarbodiimide (DIC) as coupling reagent to afford N-hydroxysuccinimido ester 8 in quantitative yield after urea byproduct filtration and trituration in petroleum ether (scheme 2, reaction f). Crude 8 is immediately used in the subsequent reaction with peptide 2 with N-methylmorpholine (NMM) as base in DMF to provide conjugate 3 in a process that takes about 2 days. The isolation of crude 3 from the reaction mixture involves neutralization to pH=6.0 with acetic acid, removal of DMF under vacuum, and dissolution of the resulting residue in 0.1 M ammonium acetate buffer.
Crude 3 is then subjected to chromatographic purification (0.1 M NaClO4/MeCN buffer). The fractions with low purity (<60%) are discarded and the fractions in the 60-95% purity range re-chromatographed (0.1% TFA water/MeCN buffer). The fractions in the 80-95% purity range are re-chromatographed under the same conditions and the fractions with purity below 80% discarded. The fractions with >96% purity and no single impurity above 1.5% are pooled and lyophilized to give clean 3 in 40.4% yield (molar basis). This chromatographic purification of 3 takes about 2 days. The lyophilized fractions are then reconstituted (in other words, a full redissolution of the solid in an appropriate solvent), in a 1:1 CH3CN/H2O mixture to generate a homogeneous lot of intermediate 3, in a process that takes about 2 days.
After filtration of some insoluble material, the filtrates are subjected to a second lyophilization to generate 3 in about 11.9% yield, in a process that takes about 2 days. While this lengthy and energy- and solvent-intensive process is quite suitable for the generation of small batches of material (˜50 g), its implementation in the manufacture of larger quantities of 3 is relatively impractical due to the very low throughput, high scale-up costs, and lengthy process time of about 10 days. There therefore exists a need to develop a process to generate large amounts of peptide-linker conjugate 3, capable of producing multi-hundred gram quantities under cGMP conditions in a time- and cost-effective manner.